Recent advances in protein engineering, chemistry, and fluorescence microscopy have resulted in improved quantitative reporters of signaling events in living cells (Zhang, J. et al., Nat. Rev. Mol. Cell. Biol., 2002, 3(12):906–18). For example, the engineering of spectrum-altered fluorescent proteins (FPs) from the Aequorea victoria Green Fluorescent Protein (GFP) has enabled simultaneous real-time measurement of multiple protein expression and localization patterns in live cells. FP-based indicators are less toxic than simple organic dyes and can respond to a wider range of biological events; they can also be targeted to subcellular compartments through genetic fusion and can be introduced into a wider variety of tissues and into intact organisms.
Although great strides have been made in FP-based indicator development, there are drawbacks in the existing technology. Existing indicators have been designed on a “custom cut”, one-at-a-time basis. They are thus currently capable of reporting only a handful of the thousands of cellular signaling state variables. Additionally, few existing FP indicators have been developed to report on the more complex cellular parameters such as enzyme activity. These “hidden” variables are implicated in every known signaling pathway, but their direct observation has not been effectively addressed by current methodology.